Mechanism of β2AR regulation by an intracellular positive allosteric modulator

Abstract

Drugs targeting the orthosteric, primary binding site of G protein-coupled receptors are the most common therapeutics. Allosteric binding sites, elsewhere on the receptors, are less well-defined, and so less exploited clinically. We report the crystal structure of the prototypic β₂-adrenergic receptor in complex with an orthosteric agonist and compound-6FA, a positive allosteric modulator of this receptor. It binds on the receptor's inner surface in a pocket created by intracellular loop 2 and transmembrane segments 3 and 4, stabilizing the loop in an α-helical conformation required to engage the G protein. Structural comparison explains the selectivity of the compound for β₂- over the β₁-adrenergic receptor. Diversity in location, mechanism, and selectivity of allosteric ligands provides potential to expand the range of receptor drugs.

Fig. 1.. Structure of the active-state T4L-β₂AR in complex with the orthosteric agonist BI-167107, nanobody 6B9, and Compound-6FA.

A, The chemical structure of Compound-6FA (Cmpd-6FA). B, Isoproterenol (ISO) competition binding with ¹²⁵I-cyanopindolol (CYP) to the β₂AR reconstituted in nanodiscs in the presence of vehicle (0.32 % dimethylsulfoxide; DMSO), Cmpd-6, or Cmpd-6FA at 32 μM. Values were normalized to percentages of the maximal ¹²⁵I-CYP binding level obtained from a one-site competition binding-Log IC₅₀ curve fit. Binding curves were generated by GraphPad Prism. Points on curves represent mean ± SEM obtained from five independent experiments performed in duplicate. C, Analysis of Cmpd-6FA interaction with the BI-167107-bound β₂AR by isothermal titration calorimetry (ITC). Representative thermogram (insert) and binding isotherm, of three independent experiments, with the best titration curve fit are shown. Summary of thermodynamic parameters obtained by ITC: binding affinity (Kd = 1.2 ± 0.1 µM), stoichiometry (N = 0.9 ± 0.1 sites), enthalpy (ΔH = 5.0 ± 1.2 kcal/mol), and entropy (ΔS =13 ± 2.0 cal/mol/deg). D, Side-view of T4L-β₂AR bound to the orthosteric agonist BI-167107, nanobody 6B9 (Nb6B9), and Cmpd-6FA. The gray box indicates the membrane layer as defined by the OPM database. e, Close-up view of Cmpd-6FA binding site. Covering Cmpd-6FA is 2F_o – F_c electron density contoured at 1.0 σ (green mesh).

Fig. 2.. Intermolecular interaction between the β₂AR and Cmpd-6FA.

A, Overview of the Cmpd-6FA binding site. The molecular surface of the β₂AR bound to Cmpd-6FA is subdivided into three surface regions: membrane embedded (green), membrane-proximal (purple), and cytoplasmic (cyan). B, C, Detailed interactions of the β₂AR bound to Cmpd-6FA. B, The side-chains of those residues that interact with the core scaffold of Cmpd-6FA are shown. The hydrogen bond is indicated by a black dashed line. C, Rotated view showing all β₂AR residues that contribute at the β₂AR–Cmpd-6FA interface.

Fig. 3.. Mechanism of allosteric activation of the β₂AR by Cmpd-6FA.

A, Superposition of the inactive β₂AR bound to the antagonist carazolol (PDB code: 2RH1) and the active β₂AR bound to the agonist BI-167107, Cmpd-6FA, and Nb6B9. Close-up view of the Cmpd-6FA binding site is shown. The residues of the inactive (yellow) and active (blue) β₂AR are depicted and the hydrogen bond formed between Asp130^3.49 and Tyr141^ICL2 in the active-state is indicated by a black dashed line. B, Topography of Cmpd-6FA binding surface on the active β₂AR (left, blue) and the corresponding surface of the inactive β₂AR (right, yellow) with Cmpd-6FA (orange sticks) docked on top. Molecular surfaces are of only those residues involved in interaction with Cmpd-6FA. Steric clash between Cmpd-6FA and the surface of inactive β₂AR is represented by a purple asterisk. C, Overlay of the β₂AR bound to BI-167107, Nb6B9, and Cmpd-6FA with the β₂AR–G_s complex (PDB code: 3SN6). The inset shows the position of Phe139^ICL2 relative to the α-subunit of G_s. D, Superposition of the active β₂AR bound to the agonist BI-167107, Nb6B9, and Cmpd-6FA (blue) with the inactive β₂AR bound to carazolol (yellow) (PDB code: 2RH1) as viewed from the cytoplasm. For clarity, Nb6B9 and the orthosteric ligands are omitted. The arrows indicate shifts in the intracellular ends of the TMs 3, 5, and 6 upon activation and their relative distances.

Fig. 4.. Gain-of-function of Cmpd-6 on the human β₁AR mutants as compared to wild-type.

A, Partial protein sequence alignment of the human β₂AR (blue highlight) and β₁AR (red highlight) that includes only the relevant locations that engage Cmpd-6FA. Those residues that interact with Cmpd-6FA and are conserved are highlighted in green. Residues that are not conserved, but also interact with Cmpd-6FA are highlighted in yellow. Residue numbers for the β₂AR and β₁AR are shown above and below the alignment, respectively. B-E, Cmpd-6 positive allosteric effect on agonist binding of B, wild-type β₂AR C, wild-type β₁AR D, L154V / L158F / R165K / R174K / G177V / L178I / T181M β₁AR mutant and E, L158F / R174K β₁AR mutant. ¹²⁵I-CYP vs. ISO competition binding was done using membrane preparations from HEK293 cells expressing the indicated receptor in the absence or presence of Cmpd-6 at 25 μM. Values were normalized to percentages of the maximal ¹²⁵I-CYP binding level obtained from a one-site competition binding-Log IC₅₀ curve fit (GraphPad Prism) of the vehicle (0.25% dimethylsulfoxide; DMSO) control data. Points on curves represent mean ± SEM obtained from four independent experiments performed in duplicate.